Download I N IN VIVO VITRO

Onderstep oort ]. 1#. R es. 37 (4), 225-228 1970
RE SEARCH NOTE
FAILURE TO DEMONSTRATE IN VITRO AS OPPOSED TO IN VIVO TRANSCRIPTION OF TH E BLUETONGUE VIRUS GENOME
D. W. VERW O E RD, Veterinary Research Institute, Onderstepoort
Thus far it has been demonstrated unequivocally
only in the case of reovirus and R D V that the doublestranded viral RNA is inactive as a template for in
z1itro protein synthesis (Shatkin, 1965; Miura & Muto,
1965). It has been shown, however, that in bluetongue
virus-infected cells a single-stranded RNA is synthesized, similar to the messenger RNA in reovirusinfected cells (Huismans, 1970). A large proportion of
this RNA is associated with polyribosomes, thus suggesting a messenger function. Furthermore, the singlestranded RNA can be fractionated by means of polyacrylamide gel-electrophoresis into fragments equivalent to the segments of the genome and in hybridization
studies it was shown to be completely homologous to
the double-stranded RNA . However, it does not show
any self-hybridization, indicating that it is a copy of
The bluetong ue virus (BTV) possesses a segmented, do uble-stranded ribonucleic acid (RNA) genome, a characteristic it shares with other members of
the Diplornavirus group (Verwoerd, Louw & Oellermann, 1970; Verwoerd, 1970). These include reovirus,
African h orsesickness virus (AH SV), wound tumor
virus (WTV), rice dwarf virus (RDV), cytoplasmic
polyhedrosis virus (CPV) and possibly a number of
other viruses of w hich the double-stranded nature of
the RNA has not yet been proved.
With a genome of this nature, the elucidation of the
enzymatic mechanisms concerned in both replication
and expression of the double-stranded RNA, poses a
problem. Attempts to demonstrate the enzyme activity
responsible for the latter process are d escribed in this
paper.
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Hours after infection
FrG. 1 Incorporation of 14C-uridine (0.02 flCifml) by BTV-infected BHK-cells in the presence of 2flgfml actinomycin D and 20 flgfml
cycloheximide ( 0 - - 0 infected cells, x· · · · x uninfected control cells).
Received 2 December 1970
225
FAILURE TO DEMONSTRATE TRANSCRIPTION OF THE BLUETONGUE VIRUS GENOME
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FrG. 2 Comparison of the electrophoretic patterns obtained on polyacrylamide gels with the hybrid product (x· · · ·x) and with labelled
double stranded BTV-RNA ( 0 - -0).
found with BTV over a wide range of conditions. These
included treatment with chymotrypsin at concentrations
ranging from zero to 500 p.gfml, pH values between
6.0 and 9.0, temperatures of incubation between 25 ° and
40 °C, ionic concentrations between 0.01M and 0.2M
and the addition of various ions, e.g. Mn +, NH4 ++
and K +. Alternative procedures were also employed
in an attempt to "activate" the enzyme, such as treatment of the virus with trypsin, pronase, Nonidet P-40,
Triton N-100, sodium dodecyl sulphate, urea, alkaline
treatment according to Lewandowski eta!. (1969), heatshock according to Borsa & Graham (1968), ultrasonification and preincubation with cytoplasmic and
nuclear extracts. In view of the relative instability of
BTV in comparison with reovirus, either one per cent
bovine serum albumin or four per cent sucrose (Howell,
Verwoerd & Oellermann, 1967) was also added in an
attempt to stabilize the virus during incubation.
_
No appreciable incorporation of 14C-uridine in the
presence of BTV was found in any of these experiments,
whereas active synthesis of RNA was present in the
reovirus controls throughout. As a result of the failure
to detect in vitro transcription with BTV, an attempt was
made to demonstrate in vivo activity. The approach was
to prevent the synthesis of any virus-induced enzyme
in infected cells by the inhibition of all protein synthesis
with high concentrations of cycloheximide. In order to
detect virus-specific RNA synthesis, the formation of
cellular RNA was concomitantly inhibited by means of
actinomycin D. Under these conditions, incorporation
of labelled uridine was regarded as viral-specific RNA
synthesis mediated by a viral enzyme.
The result of such an experiment is shown in Fig. 1.
A suspension culture of baby hamster kidney (BHK)
cells was pre-incubated for .one hour with ·actinomycin
D at a concentration of 2 f-Lgfml and cycloheximide at a
concentration of20 [Lg/ml (Watanabe, Kudo & Graham,
only one of the strands of the double-stranded RNA.
From these observations it has been concluded that the
genome of the bluetongue virus is transcribed into a
single-stranded RNA which functions as a messenger in
the synthesis of viral-specific proteins (Huismans, 1970).
The question which immediately arises is where the
enzyme responsible for the transcription process is
situated. It is unlikely that such an enzyme would be
present in the cell, where RNA-dependent RNA synthesis does not normally occur. The information for
such an enzyme could exist in the viral genome, but this
information would also have to be transcribed before
the enzyme could be synthesized in the virus-infected
cell, unless a limited portion of the genome could act
directly as messenger in the translation process. Although the latter possibility cannot be excluded completely, it does not seem very probable. The most
likely explanation is the presence of such an enzyme in
the virus particle itself.
In fact, an enzyme of this nature, provisionally
named transcriptase, has been found to be present in
reovirus (Borsa & Graham, 1968; Shatkin & Sipe,
1968; Skehel & Joklik, 1969; Levin, Mendelsohn,
Schonberg, Klett, Silverstein, Kapuler & Acs, 1970), in
CPV (Lewandowski, Kalmakoff & Tanada, 1969) and
in WTV (Black & Knight, 1970). In the case of reovirus, demonstration of in vitro enzyme activity is
dependent on prior treatment of the virions with
chymotrypsin, in order to remove the outer protein
capsid. For the other viruses, where an outer capsid is
absent, this "activation" was not found to be necessary.
However, all attempts to demonstrate in vitro RNA
synthesis with purified BTV as a source of enzyme were
unsuccessful. Using the standard incubation mixture
(Skehel & Joklik, 1969) and reovirus, purified in
exactly the same way as BTV (Verwoerd, 1969), as a
positive control, no incorporation of 14C-uridine was
226
D. W. VER \\OERD
1967). Half of the culture was then infected with purified
BTV at an input-multiplicity of 20 virions per cell.
Two hours later, H(-uridine was added to both the
infected and the non-infected control cultures at a
concentration of 0.02 J.LCijml. Duplicate samples of
2 X 10 6 cells were taken at 30 minute intervals from 2+
to 7 hours after infection and prepared forscintillatio;}
counting as previously described (Verwoerd & Huismans, 1969). The remaining cells were collected by
centrifugation and single-stranded RNA isolated by
means of phenol extraction and salt-precipitation according to procedures described elsewhere (Bellamy,
Shapiro, August & Joklik, 1967). Hybridization of the
isolated product with unlabelled double-stranded BTVR~A and analysis of the hybrid by means of polyacrylamide gel electrophoresis was carried out as described
by Huismans (1970).
The result of a hybridization experiment is shown
in Fig. 2. The pattern obtained was identical to that
obtained by Huismans for a hybrid of messenger RNA
and BTV-RNA. These results taken together clearly
indicate that a virus-specific single-stranded RNA,
capable of hybridizing with the viral genome, is synthesized in BTV-infected BHK cells at a linear rate for
at least 7 hours after infection, under conditions where
synthesis of cellular RNA and protein is inhibited.
Although the possibility of some residual protein
synthesis sufficient for the formation of a small amount
of rirus-induced enz\·me cannot be excluded, the RNA
synthesis seems to ·be dependent on a pre-existing,
actinomycin D-resistant, double-stranded RNA- dependent Rl\"A polymerase. The fact that no RNA synthesis
could be detected in non-infected control cultures and
the improbability of such an enzyme being present in
normal cells, clearly indicates the presence of an enzyme,
similar to transcriptase described for reovirus, in the
bluetongue virus particle.
The complete failure to demonstrate the activity
of such an enzyme in vitro cannot be explained at
present. It could be a reflection of the structural differences between the viruses, or could be related to the
relative instability of purified BTV. Conceivably some
cellular activity might be necessary to "uncover" or to
"activate" the enzyme. These possibilities are being
explored further.
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